The principal mission of the Clinical and Molecular Immunology Group within the Clinical Immunology Section is to perform cellular, molecular and clinical studies aimed at understanding the basis of immune tolerance. A long term objective is to develop new therapies for allograft transplantation and the treatment of autoimmune and inflammatory diseases. I. CD28 Signaling Signaling through the CD28 receptor during T cell activation exerts a profound influence on the outcome of T cell receptor (TCR) engagement. Failure to receive a costimulatory signal through CD28 results in an unresponsive state termed anergy or in T cell death; both of which contribute to the induction and maintenance of immune tolerance. Thus CD28 signaling is a critical determinant of T cell activation and is likely to be an important factor in the development of both auto- and allo- immunity. While signal transduction through the T cell receptor has been extensively characterized, the CD28 signaling pathway is poorly understood. To decipher this pathway, we've utilized a model system in which CD28 signaling is responsible for greater than 99% of T cell IL-2 production. Earlier work had shown that this CD28 dependent regulation of IL-2 expression is not at the transcriptional or translational levels but rather a consequence of increased IL-2 mRNA stability. We have shown that the AUUUA sequences within the 3' untranslated region of the IL-2 mRNA responsible for mRNA instability do not confer CD28 responsiveness. In addition, we discovered the presence of an additional mRNA instability element located within exon 3. The instability conferred by this element appears to be enhanced, rather than retarded by CD28 costimulation. Recent evidence indicates that CD28 may also negatively regulate other genes during human T cell activation, particularly the HIV coreceptor CCR5. This system that was developed to study CD28 regulation of IL-2 is now also being used to address whether post-transcriptional mechanisms play a role in the negative regulation of CCR5 in T cells. We have determined that sequences upstream of the 3'UTR, localized to exon 2 and the coding region of exon 4, are required for CD28 mediated IL-2 mRNA stabilization. Within these same regions of the IL-2 mRNA we have identified a sequence motif that may be critical for CD28 responsiveness. To test the role of this putative CD28 response element (RE) nearly a dozen mutants have been generated and stable cell lines carrying these mutations have been established. The results of these studies should be submitted for publication in the next six months. Information from these studies will assist us in the identification of RNA binding proteins that interact with cytokine mRNAs in a CD28 specific fashion. Studies carried out to identify proteins that bind the IL-2 mRNA initially focused on the 3'UTR of the mRNA but are now being extended to examine binding to the putative CD28RE. To date, constitutively expressed and TCR regulated sequence specific proteins that bind the 3'UTR have been found. One of these appears to be HuR, the mammalian homolog of a protein (ELAV) involved in the embryonic development of the neural system in Drosophila. In nonlymphoid cells HuR is associated with mRNA stabilization, however, we do not observe a change in HuR binding to the IL-2 mRNA in association with CD28 costimulation. Studies of the IL-2 mRNA 5' UTR have identified an alternative transcriptional start site for the IL-2 gene. The biological significance of this finding is being pursued. In addition to furthering our elucidation of the CD28 costimulatory pathway, these observations may have important consequences for our understanding of how IL-2 gene expression is regulated at both the transcriptional and post-transcriptional levels. The results of these studies should be submitted for publication early next year. II. Molecular Consequences of IL-2 Receptor Blockade Laboratory investigations to understand the mechanism(s) by which an antibody (HAT) against the alpha chain of the high affinity IL-2 receptor, which is used in our clinical trials, acts to ameliorate autoimmune uveitis have just completed their first phase. We found that HAT blocks the expression of multiple cytokines (both Th1 and Th2) implicated in the pathogenesis of autoimmune disease. Interestingly, HAT blocks IFN-gamma expression as effectively as an antibody against IL-12, the cytokine principally associated with the induction of IFN-gamma. We have gone on to define the mechanisms by which HAT inhibits IFN-gamma production. HAT inhibits IL-12 dependent IFN-gamma production by blocking the expression of CD40L, which itself is critical for the induction of IL-12 by antigen presenting cells. In addition, HAT can directly inhibit the production of IL-12 independent IFN-gamma from T cells by mechanisms that have not been fully elucidated. These findings have important implications for the choice of immunosuppressive regimen (e.g. HAT vs. anti- IL-12) employed in the setting of transplantation or autoimmune disease. III. Oral Tolerance Evidence from a number of labs suggests that there exists within the lymphatic tissue of the gastrointestinal tract regulatory cells that function to maintain tolerance to foreign (e.g. plant) proteins that traverse the GI tract. This has led to clinical attempts to treat autoimmune disease by feeding the putative antigen to patients. Out of necessity, these studies either utilized an animal homolog of the human antigen or limited quantities of impure recombinant human antigen; factors that possibly contributed to the inconclusive results that were obtained. Attempts to induce/reestablish systemic tolerance via the oral route might be enhanced if sufficient quantities of pure human antigen could be administered to patients. Once again, the use of DNA encoding the relevant antigen (i.e. a DNA "vaccine") may represent a potential solution to this problem as it can be produced in very large quantities to a very high purity. Recent work by our collaborator at John Hopkins has demonstrated that DNA can be effectively delivered to the GI tract via the oral route. We have found that oral administration of a human IRBP DNA vaccine appears to slightly reduce the experimental disease induced in mice by the human protein antigen administered with a very powerful adjuvant. Approaches that might better reflect the clinical situation and methods to enhance the protective effect are being investigated.